Macrophages are innate immune cells of dynamic phenotype as they can acquire distinct phenotypes and biological functions depending on the microenvironment and the metabolic state. In response to various signals, macrophages can undergo M1 activation (stimulated by TLR ligand and interferon-gamma) or M2 activation (stimulated by IL-4/IL-13, immunocomplexes, TGFbeta, and IL-10). M1 and M2 macrophages have distinct metabolic phenotypes that differ from those of resting macrophages. M1 macrophages rely on aerobic glycolysis, while M2 macrophages use fatty acid oxidation to fuel mitochondrial oxidative phosphorylation. IL-4/IL13 induced M2 macrophages polarization has an enhanced mitochondrial oxygen-consumption rate, whereas M1 has a significantly decreased oxygen-consumption rate. This indicates that macrophage phenotypes can be regulated by the different aspects of cellular metabolism. Thus, the identification of molecules and mechanisms associated with the phenotypic switch of them provides a molecular basis for macrophage-centered therapeutic strategies. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously found that GMFG-knockdown macrophages enhanced IL-4/IL-13-stimulated M2 markers and promoted iron metabolism change to iron recycling states, but its underlying molecular mechanism remains unclear. In this study, to explore the possible role of GMFG on mitochondrial function, we measured mitochondrial respiration by determining the oxygen consumption rate (OCR) and glycolytic flux in GMFG-knockdown M0 (inactivated), M1 and M2 activated bone marrow-derived macrophages using an extracellular flux analyzer. We observed that decreased basal oxygen consumption (ORC), diminished maximal respiratory capacity (MRC) and reduced cellular ATP levels in GMFG-knockdown M0 and M2 macrophages compared with control siRNA transfected macrophages. Whereas, there were no detectable changes in the rate of extracellular acidification (ECAR) in GMFG-knockdown cells. Importantly, oxygen consumption in GMFG-knockdown cells were reduced to the same level of TGFbeta-stimulated cells, but not to the level of M1 macrophages, reflecting the loss of mitochondria fitness similar to TGFbeta-stimulated M2 macrophages. Additionally, we observed a moderately increased mitochondrial ROS levels in GMFG-knockdown cells which is similar to increased levels in M2 macrophages, but significantly lower than the increased level in M1 macrophages. We did not observe decreased mitochondrial membrane potential, but found moderately increased mitochondria mass in the GMFG-knockdown cells, which may be due to the accumulation of dysfunctional mitochondria. Moreover, we found that suppressed OCR in GMFG-knockdown cells were accompanied by suppressed mitochondrial biogenesis as shown by decreased protein expression levels of mitochondrial complex I, ISCU, MnSOD2 and CuZnSOD1 by immunoblotting analysis. We also found that GMFG expression was downregulated by treatment with hydrogen peroxide at a concentration of 0.25 mM, which accompanied with increased protein levels of TfR1 and IRP1 in macrophages. Increased mROS, TfR1 and decreased MnSOD2, CuZnSOD1 in GMFG-knockdown cells can be attenuated by treatment of 5 mM NAC and 0.01 mM MitoTEMPO for 1 hour. Furthermore, we found that the remarkable decreased protein level of Dynamin-related protein 1(Drp1), which promoted mitochondria fission, in GMFG-knockdown cells. Consistent with this, confocal microscope analysis exhibited predominantly punctate mitochondria in GMFG-knockdown cells compared with the elongated tubules mitochondria in control cells. Finally, immunoblotting analysis showed that GMFG-knockdown macrophages showed an increase of phosphorylated-CREB, which might be contributed to enhanced M2 markers in GMFG-knockdown cells. Together, these results suggest that downregulation of GMFG skewing macrophages toward a M2 phenotype through suppression of mitochondrial function, which associated with impaired mitochondria fission/fusion balance. Our results indicate that GMFG plays an important role in maintaining mitochondrial function and dynamic, which is necessary for macrophage reprogramming.